Nuclear Medicine and Biology 42 (2015) 887–892

Contents lists available at ScienceDirect

Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio[ 14C]Fluciclovine (alias anti-[ 14C]FACBC) uptake and ASCT2 expression in
castration-resistant prostate cancer cells
Masahiro Ono a,b, Shuntaro Oka b,⁎, Hiroyuki Okudaira b, Takeo Nakanishi c, Atsushi Mizokami d,Masato Kobayashi e, David M. Schuster f, Mark M. Goodman f, Yoshifumi Shirakami b,g, Keiichi Kawai aaDivision of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, Ishikawa, Japan
Research Center, Nihon Medi-Physics Co., Ltd., Chiba, Japan
Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, Ishikawa, Japand
Department of Integrative Cancer Therapy and Urology, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Ishikawa, Japane
Wellness Promotion Science Center, Institute of Medical, Pharmaceutical and Health Science, Kanazawa University, Ishikawa, Japanf
Division of Nuclear Medicine and Molecular Imaging, Department of Radiology and Imaging Science, Emory University, Atlanta, GA, USAgCurrent Address: Department of Nuclear Medicine and Tracer Kinetics, Graduate School of Medicine, Osaka University, Osaka, Japanbca r t i c l e

i n f oArticle history:
Received 27 April 2015
Received in revised form 13 July 2015
Accepted 13 July 2015
Keywords:
Fluciclovine
Anti-FACBC
ASCT2Prostate cancer
CRPC
Positron emission tomographya b s t r a c t
Introduction: trans-1-Amino-3-[18F]ﬂuorocyclobutanecarboxylic acid ([18F]ﬂuciclovine, also known as anti-[18F]
FACBC), is a tracer for positron emission tomography (PET) imaging for detection of tumors such as prostate cancer (PCa). Our previous study showed that ASCT2 (Na+-dependent amino acid transporter (AAT)) mediates
ﬂuciclovine uptake in androgen-dependent PCa cells; its expression is inﬂuenced by androgen, a key hormone
in the progression of primary PCa and castration-resistant prostate cancer (CRPC). In this study, we investigated
the uptake mechanisms and feasibility of [ 18F]ﬂuciclovine for CRPC in the androgen-dependent PCa cell line
LNCaP and LNCaP-derivatives LNCaP-SF and LN-REC4.
Methods: LNCaP-SF was established after long-term cultivation of LNCaP in steroid-free conditions, and LN-Pre
and LN-REC4 were established from LNCaP inoculated in intact and castrated severe combined immunodeﬁcient
mice, respectively. Uptake and competitive inhibition experiments were performed with trans-1-amino-3ﬂuoro[1- 14C]cyclobutanecarboxylic acid ([ 14C]ﬂuciclovine) to characterize the involvement of AATs in
androgen-dependent PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LN-REC4) cell lines. AAT expression
was analyzed by Western blotting, and [14C]ﬂuciclovine uptake in androgen-dependent PCa and CRPC-like celllines were investigated in the presence or absence of dihydrotestosterone (DHT).
Results: The contribution of Na+-dependent AATs to [14C]ﬂuciclovine uptake in all cell lines was 88−98%, and[14C]ﬂuciclovine uptake was strongly inhibited by L-glutamine and L-serine, the substrates for Na+-dependent
alanine-serine-cysteine (system ASC) AATs, in the presence of Na +. DHT enhanced ASCT2 expression in
LNCaP, LN-Pre, and LN-REC4, but not in LNCaP-SF, and the responses of ASCT2 expression to DHT correlatedwith [14C]ﬂuciclovine uptake.
Conclusions: System ASC, especially ASCT2, could play a major role in [14C]ﬂuciclovine uptake into CRPC-like andandrogen-dependent PCa cells, suggesting [18F]ﬂuciclovine-PET is applicable to the detection of CRPC as well as
androgen-dependent PCa.
Advance in knowledge: [18F]ﬂuciclovine-PET may be applied for the detection of CRPC.
Implication for patient care: [18F]ﬂuciclovine-PET may permit early intervention for CRPC treatment.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction
For more than 40 years since Huggins et al. published their study [1],androgen and androgen receptor (AR) signaling have been considered
key regulators of carcinogenesis and progression of prostate cancer(PCa) [2]. In primary PCa, cancer cell proliferation depends on androgens secreted from the testis and adrenal glands. Chemical castration⁎ Corresponding author at: Research Center, Nihon Medi-Physics Co., Ltd., Sodegaura,
Chiba, 299-0266, Japan. Fax: +81 438 62 6969.
E-mail address: shuntaro_oka@nmp.co.jp (S. Oka).
http://dx.doi.org/10.1016/j.nucmedbio.2015.07.005
0969-8051/© 2015 Elsevier Inc. All rights reserved.

targeting androgens and androgen receptor (AR), known as androgendeprivation therapy (ADT), is often chosen in locally advanced PCa aswell as metastatic PCa [3], and therapeutic efﬁcacy is monitored byserum prostate-speciﬁc antigen (PSA). Although ADT is a standard
treatment, more than half of PCa patients develop castration-resistant
prostate cancer (CRPC), an acquired androgen-independent behavior,
within several years. CRPC is one of the most aggressive recurrent PCa
types and often has poor prognosis [4]. Preclinical studies using
androgen-independent LNCaP sublines [5] demonstrated the association between CRPC progression and androgen-dependent mechanisms(e.g. AR overexpression, AR mutation, AR-hypersensitive variants,

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M. Ono et al. / Nuclear Medicine and Biology 42 (2015) 887–892

alteration of intratumor androgen metabolism) as well as androgenindependent pathways (e.g. growth factor, cytokine) [6]. Therefore,
the characteristics of CRPC may shift in response to the surrounding environment. Various drugs targeting androgens and AR have been proveneffective in CRPC patients (e.g. enzalutamide [7] and abiraterone [8]). It
is believed that AR signaling remains after PCa converts to CRPC [9].
Diagnostic imaging techniques such as SPECT and PET are usefultools for detection of PCa [10]. For example, tracers such as [ 111In]
capromab pendetide [11], [ 11C]/[ 18F]-labeled choline [12,13], and[ 11C]acetate [14] are used to detect PCa. trans-1-Amino-3-[ 18F]
ﬂuorocyclobutanecarboxylic acid ([ 18F]Fluciclovine, also known asanti-[ 18F]FACBC) is a synthetic leucine-analog amino acid PET tracer
that accumulates in PCa cells [15]. A phase I clinical trial of [ 18F]
ﬂuciclovine in Japan demonstrated the favorable pharmacokinetics for
PCa detection such as slow excretion in urine and high stability [16].
In several studies, [ 18F]ﬂuciclovine detected PCa lesions including primary PCa [17], local recurrence after radical prostatectomy [15], andmetastasis in pelvic lymph nodes and bone [15,18,19]. These results
suggest that [ 18F]ﬂuciclovine is a useful PET tracer for the initial staging
and restaging of PCa patients.
[ 18F]Fluciclovine accumulation is mediated by two neutral aminoacid transporters (AATs), Na +-dependent system ASC transporters (especially ASCT2), and Na+-independent system L transporters (especially LAT1), which are upregulated in various cancers including PCa[20–22] and are thought to be involved in ﬂuciclovine uptake into prostate cancer cells [23,24]. Moreover, the afﬁnity of ﬂuciclovine for human
ASCT2 is similar to that of natural neutral amino acids such as L-alanineand L-serine [25]. We have also demonstrated that androgen enhances
ASCT2 expression, while bicalutamide, an anti-androgen drug, inhibits
androgen-induced ASCT2 expression in an androgen-sensitive cellline, LNCaP [26]. The responses of ASCT2 to androgen and bicalutamide
correlate with trans-1-amino-3-ﬂuoro[1- 14C]cyclobutanecarboxylic
acid ([ 14C]ﬂuciclovine, also known as anti-[ 14C]FACBC) uptake [26].
These results suggest that changes in ﬂuciclovine accumulation in PCa
might reﬂect changes in AAT expression and amino acid requirements
in androgen-dependent PCa cells.
Although a number of preclinical studies of PCa have been conducted, the relationship between AAT expression and [ 18F]ﬂuciclovine uptake in CRPC cells has not been addressed. If AR-related signaling
remains intact in CRPC cells, [ 18F]ﬂuciclovine might be used to detect
CRPC, because ASCT2 expression is regulated by androgen [26,27]. In
this study, we investigated the uptake mechanisms of [ 14C]ﬂuciclovine
through AATs in CRPC-like cells, and considered the feasibility of [ 18F]
ﬂuciclovine in CRPC diagnosis.
2. Materials and methods
2.1. Reagents and radioisotope-labeled tracers
All reagents were purchased from commercial suppliers (Wako Pure
Chemical Industries, Osaka, Japan; Sigma-Aldrich Japan, Tokyo, Japan;
and Nacalai Tesque, Kyoto, Japan) unless otherwise described.
The 14C-labeled tracer was used because its long half-life(5,700 years) makes it more convenient for in vitro experiments than18
F (110 min). [ 14C]ﬂuciclovine (2.09 GBq/mmol) was synthesized[23] by Sekisui Medical (Tokyo, Japan). The radiochemical purity was
conﬁrmed by thin-layer chromatography, and found to be N95%.
2.2. Cell culture
LNCaP cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The LNCaP-derived sublines (LNCaP-SF,
LN-Pre, and LN-REC4) were established and cultured as described [5].
Brieﬂy, LNCaP-SF cells, maintained in steroid-free media in vitro, proliferate only in androgen ablation conditions, but PSA mRNA was inducedin the presence of dihydroteststerone (DHT) in a concentration-

dependent manner. LN-Pre and LN-REC4 cells, which were developed
in intact and castrated severe immunodeﬁcient mice, exhibit
androgen-sensitive PSA expression and cell growth [5].
2.3. Uptake study
Uptake experiments were performed as described [28] with minor
modiﬁcations. Cells (5×104 per well) were suspended in medium andseeded in 24-well ﬂat-bottom tissue culture plates (Becton Dickinson,
East Rutherford, NJ). After 3 days, cells were washed twice with sodium
buffer (140 mM NaCl, 5 mM KCl, 5.6 mM D-glucose, 0.9 mM CaCl2, 1 mM
MgCl2, and 10 mM HEPES, pH 7.3 at 37 °C) or choline buffer (sodium
chloride was replaced with an equivalent concentration of choline chloride), and then incubated with [ 14C]ﬂuciclovine at a ﬁnal concentrationof 10 μM for 5, 15, 30, and 60 min at 37 °C in air. Uptake was stopped byremoving the tracer solution and rapidly washing the cells twice with
ice-cold buffer. The cells were lysed in 0.1 N NaOH, and radioactivity
was measured with a Tri-Carb 2910TR liquid scintillation counter(PerkinElmer, Waltham, MA) and Ultima Gold (PerkinElmer). The protein concentration of the cell lysate was determined with a VersaMax
microplate reader (Nihon Molecular Device K.K., Osaka, Japan) using
the BCA Protein Assay Kit (Thermo Fisher Scientiﬁc, Waltham, MA).
Tracer uptake is expressed as nmol/mg protein.
2.4. Competitive inhibition uptake study
Experimental procedures were performed as described [28]. Brieﬂy,
after cells were cultured in 24-well ﬂat-bottom tissue culture plates, they
were incubated in sodium or choline buffer containing 10 μM [ 14C]
ﬂuciclovine for 5 min at 37 °C in air in the presence or absence of 2 mM inhibitors. Synthetic and natural amino acids were used as inhibitors: 2aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH; for system L in theabsence of Na+); 2-(methylamino)-isobutyric acid (MeAIB; for system A,
IMINO and PAT in the presence of Na+); N-ethylmaleimide (NEM; for system y+ and LAT2/3/4 in the absence of Na+); L-glutamine (Gln; for system
A, ASCT2, B0, N, y+L, L, and B0,+ in the presence of Na+); L-serine (Ser; forsystem A, ASCT1/2, B0, N, y+L, asc, L and B0,+ in the presence of Na+).
[ 14C]Fluciclovine uptake in sodium and choline buffers without inhibitors was normalized to 100%, and the inhibitory effects of synthetic
and natural amino acids on [ 14C]ﬂuciclovine uptake were calculated as apercentage of the control in each buffer.
2.5. Western blotting
After cells were lysed in lysis buffer (Cell Signaling Technology
Japan, Tokyo, Japan) according to manufacturer protocols, 10 μg total
protein was loaded in Any-kD PROTEAN TGX precast gels (Bio-Rad, Hercules, CA) and transferred to PVDF membrane using Transblot Turbo(Bio-Rad). The membranes were incubated in ImmunoBlock (DSPharma biomedical, Osaka, Japan) for 60 min at room temperatureand then incubated with primary antibodies overnight at 4 °C as follows: rabbit anti-ASCT2 (D7C12) monoclonal antibody (1:1000, Cell
Signaling Technology Japan); rabbit anti-androgen receptor pAb (ChIP
grade, 1:2000; Abcam, Cambridge, MA); goat anti-actin pAb (C-11)(1:5000, Santa Cruz Biotechnology, Santa Cruz, CA). The next day, themembranes were washed three times (10 min each) with Trisbuffered saline containing 0.1% Tween-20 (TBS-T). Then, membranes
were incubated with anti-rabbit or anti-goat secondary antibody conjugated to horseradish peroxidase (1:10000, Sigma-Aldrich, St. Louis, MO)
for 60 min at room temperature. After washing three times with TBS-T,
proteins were detected with ECL Prime detection reagent (GEHealthcare Japan, Tokyo, Japan) then chemiluminescence was developed using Hyperﬁlm ECL (GE Healthcare Japan), Hi-Rendol, and HiRenﬁx solution (FUJIFILM, Tokyo, Japan). The protein concentration of
the cell lysate was determined as described in Section 2.3. The molecular mass of each protein band was determined according to theM. Ono et al. / Nuclear Medicine and Biology 42 (2015) 887–892

3. Results

manufacturer's product information sheet. Band density was quantitatedusing ImageJ 1.42 software (National Institutes of Health, Bethesda, MD),and each density ratio was normalized to the loading control (actin).

3.1. Comparison of [ 14C]ﬂuciclovine uptake between androgen-dependent
PCa and CRPC-like cell lines

2.6. Flow cytometry

We compared [ 14C]ﬂuciclovine uptake in androgen-dependent PCa(LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LN-REC4) cell lines.
As shown in Fig. 1A, total [ 14C]ﬂuciclovine uptake did not signiﬁcantly
differ between androgen-dependent PCa and CRPC-like cell lines, except between LN-Pre and LN-REC4 cells. The uptake of LNCaP, LNCaPSF, LN-Pre, and LN-REC4 was as follows: 2.5±0.5, 2.4±0.3, 2.1±0.5,
and 2.6±0.4 nmol/mg protein at 5 min; 6.4±1.2, 6.0±0.9, 5.4±1.2,
and 6.7±1.0 nmol/mg protein at 15 min; 8.5±1.8, 9.6±1.4, 7.5±1.4,
and 9.7±1.4 nmol/mg protein at 30 min; and 10.1±2.7, 12.2±1.6, 9.6±2.2, and 12.1±1.7 nmol/mg protein at 60 min, respectively. The contribution of Na+-dependent AATs to [ 14C]ﬂuciclovine uptake was 88−
98% of total uptake of [14C]ﬂuciclovine in all cell lines; the contributionof Na +-independent AATs to [ 14C]ﬂuciclovine uptake was minimal(2–12%), although Na +-independent [ 14C]ﬂuciclovine uptake in
LNCaP-SF was substantially higher than in other cell lines (Fig. 1B).
These results suggest Na +-dependent AATs are largely involved in[ 14C]ﬂuciclovine uptake into LNCaP and its derivative sublines, regardless of androgen responsiveness.

Experiments were performed as described elsewhere [24], with minor
modiﬁcations. Cells were ﬁxed in 90% methanol with Ca 2+/Mg2+-freephosphate-buffered saline for 30 min on ice. Then, the ﬁxed cells weresuspended in 0.1% Triton X-100 dissolved in Ca2+/Mg2+-free HBSS with
0.5% (w/v) bovine serum albumin and 0.5 mM ethylenediaminne
tetraacetic acid for 15 min on ice for permeation of the membrane. This
step was followed by immunostaining with anti-ASCT2 primary antibody,(which recognizes the intracellular epitope, described in Section 2.5)
for 30 min on ice. Then, the cells were stained with phycoerythrin(PE)-labelled goat anti-rabbit secondary antibody for 30 min on ice.
Rabbit monoclonal antibody (DA1E) IgG (Cell Signalling Technology
Japan) was used as isotype control. Data were acquired using a
FACSCalibur ﬂow cytometer (Becton Dickinson) and analyzed with
WinMDI 2.8 software (The Scripps Institute).
2.7. Dihydrotestosterone (DHT) exposure
Cells were suspended in DMEM with 5% Charcoal/Dextran-treated
FBS (CSS; Thermo Fisher Scientiﬁc) and seeded in 6-well ﬂat-bottom tissue culture plates (5×10 5 per well). The following day, DHT dissolved indimethylsulfoxide (DMSO) was added to each well and incubated forthree days (ﬁnal concentration of DHT: 10 nM, DMSO: 0.1%). The control cells were treated with DMSO without DHT at the same concentration. Exposure conditions including the ﬁnal concentration of DHT andincubation time were determined based on the alteration of ASCT2mRNA expression in response to DHT in LNCaP as measured by PCR(data not shown). The uptake experiment (uptake buffer, sample preparation, and measurement of protein concentration) was conducted as
described in Section 2.3.

3.2. Competitive inhibition of transport in [ 14C]ﬂuciclovine in androgendependent PCa and CRPC-like cell lines
To estimate the involvement of AATs in [14C]ﬂuciclovine uptake into
androgen-dependent PCa and CRPC-like cell lines, competitive inhibition experiments were performed with natural/synthetic amino acid inhibitors (Fig. 2A–D). Gln and Ser strongly inhibited [ 14C]ﬂuciclovinetransport in the presence of Na+ (9–22% of control), and BCH moderately inhibited [14C]ﬂuciclovine uptake in choline buffer (16% of control in
LNCaP-SF, 75–84% of control in other cells). In contrast, [ 14C]ﬂuciclovine
uptake was not inhibited by MeAIB in sodium buffer or by NEM in choline buffer in all cell lines. As described in Section 3.1, the contribution of
Na +-independent (i.e. system L AATs-mediated) uptake for [ 14C]
ﬂuciclovine in the PCa cell lines used in this study was 12% at most. Considering our previous reports on ﬂuciclovine recognition [23–25], theseresults suggest systems ASC, especially ASCT2, mediates ﬂuciclovinetransport into CRPC-like cells as well as androgen-dependent PCa cells(see Section 2.4. for the characteristics of inhibitors for AATs).

2.8. Data and statistical analysis
All data are presented as the mean±standard deviation (SD). Statistical analyses were performed in SAS software version 5.0 (SAS InstituteJapan, Tokyo, Japan) using the two-tailed unpaired t-test or Dunnett's
multiple comparison tests. Pb0.05 was considered signiﬁcant.

A

B

Total uptake

Na+-independent uptake(in Sodium buffer)(in Choline buffer)

*
10

5

0
0

15

*
nmol / mg protein

nmol / mg protein

15

*
*
20

40

Time (min)

889

60

LNCaP

3

LNCaP-SF
2

LN-Pre

1
0

#
0

#

#
20

#
40

LN-REC460

Time (min)Fig. 1. (A) Time-course of total [14C]ﬂuciclovine uptake into PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LN-REC4) cells in sodium buffer over 60 min. Each point represents themean±SD (n=9). *Pb0.05 between LN-Pre and LN-REC4. (B) Time-course of Na+-independent [14C]ﬂuciclovine uptake into PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LNREC4) cells in choline buffer over 60 min. Each point represents the mean±SD (n=6). #Pb0.05 between LNCaP and LNCaP-SF.

LNCaP-SF

150
(%)

150
(%)

100

100

50

50

A

LN-REC4B

LNCaP

LN-PreA

LNCaP-SF

M. Ono et al. / Nuclear Medicine and Biology 42 (2015) 887–892

LNCaP

890

ASCT2 (70kDa)
AR (110kDa)

0*0control+Gln+Ser+MeAIB
control+BCH+NEM

Sodium buffer

control+Gln+Ser+MeAIB
control
+BCH
+NEM

0

0

Choline buffer

Fig. 2. Competitive inhibition percentages of [14C]ﬂuciclovine uptake for 5 min into(A) LNCaP (PCa), (B) LNCaP-SF (CRPC-like), (C) LN-Pre (PCa) and (D) LN-REC4 (CRPClike) cells. Cells were incubated in sodium buffer (white bar) or choline buffer (blackbar) with or without the indicated inhibitors (2 mM). [14C]Fluciclovine uptake without inhibitors (control) in sodium or choline buffer was normalized to 100%. Each value represents the mean±SD (n=4–18). *Pb0.05 vs. control in sodium buffer, #Pb0.05 vs. control
in choline buffer.

3.3. Expression of ASCT2 in androgen-dependent PCa and CRPC-like celllines
Protein expression of ASCT2 and AR in androgen-dependent PCa and
CRPC-like cell lines was analyzed by Western blotting (Fig. 3). As shownin Fig. 3A–B, ASCT2 expression in CPRC-like cells, especially in LNCaP-SF,
was lower than in androgen-dependent PCa cells (Pb0.01 between
LNCaP and LNCaP-SF; P=0.011 between LN-Pre and LN-REC4). In addition, the protein expression of AR was higher in LNCaP-SF cells, but
tended to decrease in LN-REC4 cells relative to that in the parental celllines (P=0.042 vs. LNCaP, and P=0.055 vs. LN-Pre, respectively)(Fig. 3A–B). Furthermore, the same tendency of ASCT2 expression wasobserved by ﬂow cytometry (Fig. 3C).

1

0
LN-REC4* *#LN-Pre* *1

2

LNCaP-SF

50

50

2

*

3

LNCaP

#

Density ratio (per actin)

100

LN-REC4

#

100

AR

3

LN-Pre

150
(%)

150
(%)

ASCT2

B

LNCaP-SF

LN-REC4

Density ratio (per actin)

control+Gln+Ser+MeAIB
control
+BCH
+NEM

D

LN-Preactin (44kDa)

control+Gln+Ser+MeAIB
control
+BCH
+NEM

0

C

#

LNCaP

* *

* *

C
LNCaP
20.3±1.3

LNCaP-SF

LN-PreLN-REC45.7±0.1

17.7±0.2

13.1±0.1

Fig. 3. Protein expression proﬁles of PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and
LN-REC4) were conﬁrmed by Western blotting (A, B) and ﬂow cytometry (C). In Western
blotting (A), each cell lysate (10 μg/lane) was fractionated on SDS-PAGE and incubatedwith the indicated antibodies. Actin was used as a loading control. Band density ratios(B) are shown as (the band density of each protein)/(the band density of actin). Eachratio represents the mean±SD (n=3, *Pb0.05 vs. LNCaP, #Pb0.05 vs. LN-Pre). Results of
ﬂow cytometry analysis (C), with the ﬂuorescence intensity (X-axis) and cell number(Y-axis) shown as histograms. The values are the T/N ratio calculated from the mean ﬂuorescence intensity of ASCT2 expression (closed histogram: T) and isotype control (openhistogram: N), respectively (n=3).

(n=8–9, P=0.025); and 5.9±0.2 and 7.8±0.3 nmol/mg protein in LNREC4 (n=8, Pb0.01), respectively. These results suggest that the alteration of [ 14C]ﬂuciclovine uptake correlates with the degree of ASCT2
protein expression in LNCaP, LN-Pre, and LN-REC4 cell lines.

3.4. Responses of ASCT2 expression to DHT in androgen-dependent PCa and
CRPC-like cell lines

4. Discussion

ASCT2 expression in response to DHT was investigated to characterize androgen dependency in CRPC-like cell lines. DHT enhanced ASCT2
expression 2.8-fold in LNCaP, 2.2-fold in LN-Pre, and 1.7-fold in LNREC4, but was unaltered in LNCaP-SF cells (Fig. 4A–B).
Finally, we investigated the effect of DHT on [14C]ﬂuciclovine uptake
in androgen-dependent PCa and CRPC-like cell lines. [ 14C]Fluciclovine
uptake tended to be lower in CRPC-like cells than in androgendependent PCa (Fig. 5). Further [ 14C]ﬂuciclovine uptake was increased
by DHT stimulation (1.2–1.3 fold over the controls) in LNCaP, LN-Pre,
and LN-REC4, but not LNCaP-SF cells. The uptake without and with
DHT was as follows: 8.2±0.8 and 9.6±1.5 nmol/mg protein in LNCaP(n=9, P=0.032); 5.9±0.5 and 6.4±0.8 nmol/mg protein in LNCaP-SF(n=12, P=0.092); 7.0±0.5 and 8.1±1.1 nmol/mg protein in LN-Pre

This is the ﬁrst study to investigate the differences between
androgen-dependent PCa and CRPC-like cells in the context of
ﬂuciclovine uptake, AAT involvement with inhibitors, and androgen dependency in vitro.
We previously reported that Na +-dependent AATs, especially
ASCT2, mediate [ 14C]ﬂuciclovine transport in androgen-dependent
PCa cells [23,24]. In the present study, total uptake of [ 14C]ﬂuciclovine
and inhibition proﬁles was similar in CRPC-like cells and androgendependent LNCaP cells under normal culture conditions (Figs. 1 and2), suggesting that [18F]ﬂuciclovine-PET can be used to visualize lesions
in patients with androgen-dependent PCa as well as CRPC [16,19,29].
As shown in Fig. 3, ASCT2 expression in CRPC-like cells was lower
than that in the parent cells, but the inhibition proﬁle in the presenceM. Ono et al. / Nuclear Medicine and Biology 42 (2015) 887–892

A

891

B
LNCaP

(DHT)

LNCaP-SF

LN-PreLN-REC4- - - +++ - - - +++ - - - +++ - - - +++

ASCT2
actin

Density ratio(ASCT2/actin)

4

*

CSS
DHT

3

*

2

*

1

LN-REC4

LN-PreLNCaP-SF

LNCaP

0Fig. 4. The effect of androgen on ASCT2 protein expression in PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LN-REC4) cells. Cells were incubated with steroid-free media (CSS) for
24 h, and then CSS without DHT (white bar) or with DHT (black bar) was exposed for 72 h. (A) Total ASCT2 protein expressions were analyzed using Western blotting (10 μg/lane). Theband of actin is shown as a loading control. (B) The ASCT2 expression ratio were calculated as (the band density of ASCT2)/(the band density of actin). Each ratio represents the mean±SD(n=3). *Pb0.05 vs. CSS.

of Gln and Ser (Fig. 2) was almost same between parent and CRPC-likecells. This discrepancy could be attributed to the binding sites of
ASCT2 in CRPC-like cells and their parent cells being saturated, regardless of the expression level, because the concentration of inhibitors(2 mM) used in this experiment was approximately 100-times the Km
value of ASCT2 (Km value of Gln 23.8 μM, Ser 18.8 μM) [30]. We also believe that Gln partially inhibited LAT1 activity in LNCaP-SF because LAT1
recognizes Gln with low afﬁnity (Km=1.64 mM) [31]. In the case of
LNCaP-SF, ASCT2 as well as LAT1 (only slightly) may be involved intheir observation because the expression of ASCT2 did not disappear
completely. Similarly, in the case of LN-REC4, ASCT2 may be involvedin their inhibition because ASCT2 activity was strongly inhibited by
Gln/Ser because of the high concentration of the inhibitors. Furthermore,considering substrate recognition, we suggest that not only ASCT2 but
also other sodium-dependent AATs, such as system N, may contributeto ﬂuciclovine uptake in CRPC-like cells because Gln and Ser are also substrates of SNAT5 [24]. Another possible explanation for this could be that
post-translational modiﬁcations of ASCT2, such as glycosylation level[32] and interaction with scaffold protein [33], may control the transport
function of ASCT2. Although there are some discrepancies between our
results and the characteristics of each cell line, we believe the molucularmechanisms revealed here could be beneﬁcial for future research projects because several mechanisms including androgens, growth factors,
and/or cytokines are thought to be involved in CRPC progression [5].
ASCT2 expression is upregulated in various tumors [34], and androgen regulates ASCT2 expression as well as cell growth [26]. Clinicalreports suggest ASCT2 expression decreases gradually with PCa

12

*

*

10

nmol / mg protein

CSS

8

DHT

*

6
4
2
0
LNCaP LNCaP-SF

LN-PreLN-REC4

Fig. 5. The effect of androgen on [14C]ﬂuciclovine uptake over 5 min in PCa (LNCaP and LNPre) and CRPC-like (LNCaP-SF and LN-REC4) cells. Cells were incubated in steroid-free
media (CSS) for 24 h, and then in CSS without DHT (white bar) or with DHT (black bar)for 72 h. Each value represents the mean±SD (n=8–12).*Pb0.05 vs. CSS.

progression from normal prostate to primary PCa, to PCa with ADT.
Moreover, ASCT2 expression in CRPC is maintained as it is in PCa withADT [27,35]. Our results support this observation: ASCT2 expression
gradually decreased with increasing androgen independency, i.e.
LNCaP (androgen-dependent PCa) N LN-REC4 (androgen-dependent
CRPC-like) N LNCaP-SF (androgen-independent CRPC-like). In addition,[ 14C]ﬂuciclovine uptake correlated with ASCT2 expression in the
androgen-dependent PCa and CRPC-like cells in the absence of androgen, mimicking ADT (Figs. 4 and 5). These results suggest that [ 18F]
ﬂuciclovine-PET reﬂects the androgen-sensitivity of PCa and is usefulfor monitoring tumor response to therapy in patients with androgendependent PCa and in CRPC patients treated with alternative antiandrogen therapy [36] or second generation anti-androgen drugs [7,8].
Wang et al. [37] reported an inverse relationship between LAT1 andLAT3 gene expression during the progression from primary PCa to CRPC.
To apply their putative scheme to our study, ASCT2 and LAT3 expression
is stimulated by androgen signaling in androgen-dependent PCa, andthe mammalian/mechanistic target of rapamycin (mTOR) pathway isactivated by the intracellular abundance of leucine and glutamine,transported by LAT3 and ASCT2, respectively. In contrast, ADT would
suppress LAT3 and ASCT2 expression in the primary PCa, leading to inactivation of mTOR due to a shortage of intracellular leucine and glutamine [37]. The activated transcription factor-4 (ATF4) then upregulates
LAT1 expression, followed by restoration of intracellular leucine [37]. As
a result, mTOR signaling is restored and the PCa cells recur as a CRPC.
AAT expression is also related to the cancer microenvironment in interesting ways. Intratumoral cell density generally increases with tumor
progression, and the transport activity of system ASC, but not of system
L, decreases with increasing cell density [38]. We recently demonstrated
that [ 18F]ﬂuciclovine accumulation is positively correlated with celldensity in a rat glioma model [39]. In addition, the pH in progressive
tumor tissue is comparatively acidic [40], and the transport activities
of [ 14C]ﬂuciclovine by Na+-dependent AATs including system ASC are
diminished, whereas the activity of Na+-independent AATs including
system L increased under low pH conditions [24]. Thus, the contributionof Na +-independent AATs for [18F]ﬂuciclovine in CRPC may increase in
the PCa microenvironment. As also shown in Fig. 2B, LNCaP-SF will be
the more suitable CRPC model for proving the mechanism of [ 18F]
ﬂuciclovine uptake because the contribution of LAT1 increases with increasing malignancy [21,22,37]. If our hypothesis and the proposal of
Wang et al. are correct, [ 18F]ﬂuciclovine transport is mediated by
ASCT2 during early PCa growth (e.g. androgen-dependent PCa); then,the contribution of LAT1 gradually increases after tumor progression(e.g. CRPC) because ASCT2 and LAT1 recognize ﬂuciclovine [23–25]. If
so, [ 18F]ﬂuciclovine-PET could be a feasible PET tracer for detecting all
stages of PCa progression. Further investigation is needed to assess theclinical application of these ﬁndings in CRPC patients.

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M. Ono et al. / Nuclear Medicine and Biology 42 (2015) 887–892

5. Conclusion
System ASC, especially ASCT2, probably determine [ 18F]ﬂuciclovine
uptake into CPRC-like as well as androgen-dependent PCa cells, suggesting that [ 18F]ﬂuciclovine-PET is useful for detection of CRPC aswell as androgen-dependent PCa.
Conﬂict of interest
Masahiro Ono, Shuntaro Oka, Hiroyuki Okudaira, and Yoshifumi
Shirakami are employees of Nihon Medi-Physics Co., Ltd. Mark M. Goodman and Emory University have patent rights for [ 18F]ﬂuciclovine andare eligible to receive royalties on [ 18F]ﬂuciclovine from Nihon MediPhysics Co., Ltd. David M. Schuster, Mark M. Goodman and Keiichi
Kawai have an ongoing research collaboration with Nihon MediPhysics Co., Ltd. Takeo Nakanishi, Atsushi Mizokami and Masato
Kobayashi have no conﬂict of interest.
Acknowledgments
The authors express their appreciation to Ms. Sachiko Naito (Nihon
Medi-Physics Co., Ltd.) for performing the cell culture, and to Dr.
Shunsuke Meike and Ms. Miho Ikenaga (Nihon Medi-Physics Co., Ltd.)for their helpful assistance with the Western blotting experiments.
The authors also thank Dr. Kodai Nishi (Nagasaki University) and Ms.
Asuka Mizutani (Kanazawa University) for helpful discussion.
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